Methods and compositions for single chain variable region ENOX2 antibodies for cancer detection and diagnosis

ABSTRACT

Cancers of different cellular or tissue origins express different ENOX2 cancer isoforms or combinations of isoforms and shed these proteins into the circulation. Herein are disclosed methods both for cancer detection and diagnosis of particular origin, based on the patterns and molecular weights of the isoforms which allow the identification of the cell type and or tissue of origin of the neoplasm. Relative ENOX2 amounts are proportional to tumor burden and provide a reliable measure of response to therapy and disease progression. Also provided is the amino acid sequence to which the scFv antibodies bind as the molecular basis for the specificity of the test.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. application Ser. No.14/229,091, which is a continuation of International Patent ApplicationNo. PCT/US2012/059141, filed Oct. 5, 2012 which claims the benefit ofU.S. Provisional Application No. 61/543,931, filed Oct. 6, 2011, each ofwhich is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The field of this invention is the area of protein biochemistry, inparticular, as related to the diagnosis of neoplastic and diseasedcells, as specifically related to particular isoforms of a cell surfacemarker characteristic of neoplasia in general and with specific patternsof protein expression indicative of specific types of cancer. Detectionof the particular isoforms is by use of specific antibodies.

There is a unique, growth-related family of cell surface hydroquinone orNADH oxidases with protein disulfide-thiol interchange activity referredto as ECTO-NOX proteins (for cell surface NADH oxidases) (Morre, 1998.Plasma Membrane Redox Systems and Their Role in Biological Stress andDisease (Asard, H., Berczi, A. and Caubergs, R. J., Eds) pp. 121-156,Kluwer Academic Publishers, Dordrecht, Netherlands; Morre and Morre,2003. Free Radical Res. 37: 795-805). One member of the ECTO-NOX family,designated ENOX2 (for tumor associated) is specific to the surfaces ofcancer cells and the sera of cancer patients (Morre et al., 1995. Proc.Natl. Acad. Sci. 92: 1831-1835; Bruno et al., 1992. Biochem. J. 284:625-628). The presence of the ENOX2 protein has been demonstrated forseveral human tumor tissues (mammary carcinoma, prostate cancer,neuroblastoma, colon carcinoma and melanoma) (Cho et al., 2002. CancerImmunol. Immunother. 51: 121-129); and serum analysis suggest a muchbroader association with human cancer (Morre, et al., 1997. Arch.Biochem. Biophys. 342: 224-230; Morre and Reust, 1997. J. Bioenerg.Biomemb. 29: 281-289).

NOX proteins are ectoproteins anchored in the outer leaflet of theplasma membrane (Morre, 1965. Biochim. Biophys. Acta 1240: 201-208; FIG.1). As is characteristic of other examples of ectoproteins (sialyl andgalactosyl transferase, dipeptidylamino peptidase IV, etc.), the NOXproteins are shed. They appear in soluble form in conditioned media ofcultured cells (Cho et al., 2002. Cancer Immunol. Immunother. 51:121-129) and in patient sera (Morre, et al., 1997. Arch. Biochem.Biophys. 342: 224-230; Morre and Reust, 1997. J. Bioenerg. Biomemb. 29:281-289). The serum form of ENOX2 from cancer patients exhibits the samedegree of drug responsiveness as does the membrane-associated form.Drug-responsive ENOX2 activities are seen in sera of a variety of humancancer patients, including patients with leukemia, lymphomas or solidtumors (prostate, breast, colon, lung, pancreas, ovarian, liver) (Morre,et al., 1997. Arch. Biochem. Biophys. 342: 224-230; Morre and Reust,1997. J. Bioenerg. Biomemb. 29: 281-289). An extreme stability andprotease resistance of the ENOX2 protein (del Castillo-Olivares et al.,1998. Arch. Biochem. Biophys. 385: 125-140) may help explain its abilityto accumulate in sera of cancer patients to readily detectable levels.In contrast, no drug-responsive NOX activities have been found in thesera of healthy volunteers (Morre, et al., 1997. Arch. Biochem. Biophys.342: 224-230; Morre and Reust, 1997. J. Bioenerg. Biomemb. 29: 281-289)or in the sera of patients with disorders other than neoplasia.

While the basis for the cancer specificity of cell surface ENOX2 was notpreviously determined, the concept was supported by several lines ofevidence. A drug responsive ENOX2 activity has been rigorouslydetermined to be absent from plasma membranes of non-transformed humanand animal cells and tissues (Morre et al., 1995. Proc. Natl. Acad. Sci.92: 1831-1835). The ENOX2 proteins lack a transmembrane binding domain(Morre, et al., 2001. Arch. Biochem. Biophys. 392: 251-256) and arereleased from the cell surface by brief treatment at low pH (delCastillo-Olivares et al., 1998. Arch. Biochem. Biophys. 358: 125-140). Adrug-responsive ENOX2 activity has not been detected in sera fromhealthy volunteers or patients with diseases other than cancer (Morre,et al., 1997. Arch. Biochem. Biophys. 342: 224-230; Morre and Reust,1997. J. Bioenerg. Biomemb. 29: 281-289).). Several ENOX2 antisera haveidentified the immunoreactive band at 34 kDa (the processed molecularweight of one of the cell surface forms of ENOX2) with Western blotanalysis or immunoprecipitation when using transformed cells and tissuesor sera of cancer patients as antigen source (Cho et al., 2002. CancerImmunol. Immunother. 51: 121-129; Morre, et al., 2001. Arch. Biochem.Biophys. 392: 251-256; Chueh et al., 2002. Biochemistry 44: 3732-3741).The immunoreactive band at 34 kDa is absent with western blot analysisor immunoprecipitation when using transformed cells and tissues or serafrom healthy volunteers or patents with disorders other than cancer (Choet al., 2002. Cancer Immunol. Immunother. 51: 121-129; Morre, et al.,2001. Arch. Biochem. Biophys. 392: 251-256; Chueh et al., 2002.Biochemistry 44: 3732-374). These antisera include a monoclonal antibody(Cho et al., 2002. Cancer Immunol. Immunother. 51: 121-129),single-chain variable region fragment (scFv) which reacts with the cellsurface NADH oxidase from normal and neoplastic cells, polyclonalantisera made in response to expressed ENOX2 (Chueh et al., 2002.Biochemistry 44: 3732-374) and polyclonal peptide antisera to theconserved adenine nucleotide binding region of ENOX2 (Chueh et al.,2002. Biochemistry 44: 3732-374).

ENOX2 cDNA has been cloned (GenBank Accession No. AF207881; 11; U.S.Patent Publication 2003-0207340 A1). The derived molecular weight fromthe open reading frame was 70.1 kDa. Functional motifs include a quinonebinding site, an adenine nucleotide binding site, and a cysteine pair asa potential protein disulfide-thiol interchange site based onsite-directed mutagenesis (Chueh et al., 2002. Biochemistry 44:3732-374). Based on available genomic information (Bird, 1999. Directsubmission of human DNA sequence from clone 875H3 (part of APK1 antigen)to GenBank database at NCBI) the ENOX2 gene is located on chromosome X,and it is comprised of multiple exons (thirteen). It is known that thereare a number of splice variant mRNAs and proteins expressed.

The hybridoma cell line which produces the tumor NADH oxidase-specificmonoclonal antibody MAB 12.1 was deposited with the American TypeCulture Collection, Manassas, Va., 20108 on Apr. 4, 2002, under theterms of the Budapest Treaty. This deposit is identified by AccessionNo. ATCC PTA-4206. The deposit will be maintained with restriction inthe ATCC depository for a period of 30 years from the deposit date, or 5years after the most recent request, or for the effective life of thepatent, whichever is longer, and will be replaced if the deposit becomesnon-viable during that period. This monoclonal antibody is described inU.S. Pat. No. 7,053,188, issued May 30, 2006, which is incorporated byreference herein.

Because cancer poses a significant threat to human health and becausecancer results in significant economic costs, there is a long-felt needin the art for an effective, economical and technically simple system inwhich to assay for the presence of cancer.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method for the analysis of a biologicalsample for the presence of particular isoforms of the pan-cancer antigenknown as ENOX2 (for tumor-specific NADH oxidase). The present methodentails 2-dimensional gel electrophoresis and immunoblotting using anantibody specific for the pan-cancer ENOX2 antigen and the variousisoforms which characterize particular types of cancers. As specificallyexemplified, about 30 μL of sera are loaded for analysis.

The present invention provides a method for the detection of particularcancer-specific serum or plasma ENOX2 isoforms as indicators of thepresence of cancer and of the cell type or tissue of cancer origin(breast, ovarian prostate, etc.). ENOX2 isoforms are identified on thebasis of their molecular mass and isoelectric point with detection usinga ENOX2-specific monoclonal antibody (MAB) (U.S. Pat. No. 7,053,188),using single chain variable region (ScFv) fragment which recognizes allcell surface NOX proteins (both age-related, normal cell and neoplasiaspecific NADH oxidase) or using polyclonal sera raised against ENOX2.The ECTO-NOX proteins are first enriched and concentrated from abiological sample, desirably a serum sample, by binding tonickel-agarose and then eluting. After release of the proteins from thenickelagarose by vortexing, the proteins are separated in the firstdimension by isoelectric focusing and in the second dimension bypolyacrylamide gel electrophoresis. As specifically exemplified herein,the isoelectric focusing step is over a pH range from 3 to 10, and sizeseparation is over a 10% polyacrylamide gel. Most of the cancer-specificENOX2 isoforms exhibit isoelectric points in a very narrow range betweenpH 3.9 and 6.3 but differ in molecular weight from 34 to 136 kDa. In the2D gel system specifically exemplified, the cancer-specific isoforms arelocated in Quadrants I (relatively high molecular weight material) andIV (lower molecular weight material, notably the range of 30 to 30 kDa.IgG heavy chains (Quadrant II and IgG light chains (Quadrant III) crossreact with the ScFv antibody and along with reference proteins at 53 and79 to 85 kDa serve as loading controls. The absence of all ENOX2isoforms indicates the absence of cancer. The presence of an ENOX2isoform indicates the presence of cancer. The particular molecularweight present in a serum sample or a particular combination of isoformsprovides an indication of the cell type or tissue of origin of thecancer. The method not only determines cancer presence, but also themethod of the present invention provides diagnostic informationconcerning the tissue of origin. At present there are no other pancancer (all forms of human cancer) tests with these particularcapabilities.

The present invention provides a method for determining neoplasia in amammal, including a human, said method comprising the steps of detectingcancer presence, in a biological sample. The present invention furtherprovides additional information for assessment of neoplasia, including ameasure of tumor burden, for example in serum, plasma, urine, saliva orin biopsy material.

Also within the scope of the present invention are particular isoformsof ENOX2 associated with specific (primary) cancers. ENOX2 proteins withapparent molecular weights of about 64, 66 and/or 68 kDa. pH 4.5 areassociated with breast cancer. ENOX2 protein of 52 kDa, pH 4.3 isassociated with small cell lung cancer. ENOX2 proteins of 52 and 80 kDa,pH 4.1 and 4.2 characterize ovarian cancer. ENOX2 isoforms of about 75kDa, pH 6.3 are associated with prostate cancer. An ENOX2 protein ofabout 94 kDa, pH 5.4 is associated with cervical cancer. ENOX2 proteinsof about 34 and 52 kDa, pH 4.3 and 3.9 are characteristic of coloncancer. An ENOX2 isoform of 54 kDa, pH 5.1 is associated with non smallcell lung cancer. Where a patient is suspected of having cancer, abiological sample, advantageously a serum sample can be prepared, andthe 2D gel electrophoresis/immunological analysis of the presentinvention can be undertaken. Positive results are indicative of thepresence of cancer, and the detection of characteristic proteins allow apresumption as to the primary incidence of cancer in that patientaccording to the association of particular protein(s) with particularcancer origins, as set forth above.

The methods of the present invention can also be applied to evaluateresponse to therapy, with decreasing amounts of NOX isoform(s)reflecting successful treatment, as well as early detection of recurrentdisease (reflected increased or reappearance of ENOX2-specific isoforms.

Specifically the invention teaches the structure, epitopecharacterization, binding affinity, specificity of the antigens to whichthe recombinant detecting antibodies must bind.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the utility of the ENOX2 familyof cancer-specific, cell surface proteins for early diagnosis and earlyintervention of cancer. Cancer site-specific transcript variants ofENOX2 are shed into the serum to permit early detection and diagnosis.The ENOX2 proteins of origin at the cell surface act as terminaloxidases of plasma membrane electron transport functions essential tothe unregulated growth of cancer. When the ENOX2 proteins are inhibited,as for example through EGCg/Capsicum synergies, the unregulated growthceases and the cancer cells undergo programmed cell death (apoptosis).

FIGS. 2A-2B provide a 2-Dimensional gel/western blot of ENOX2 transcriptvariants comparing pooled non-cancer (FIG. 2A) and pooled cancerrepresenting major carcinomas plus leukemias and lymphomas (FIG. 2B)patient sera. The approximate location of unreactive (at background)albumin is labeled for comparison. ENOX2 reactive proteins arerestricted to quadrants I and IV. Detection use recombinant scFv-S(LysGluThrAlaAlaAlaLysPheGluArgGln HisMetAspSer; SEQ ID NO:8) antibodylinked with alkaline phosphatase. The approximately 10 ENOX2 transcriptvariants of the pooled cancer sera are absent from non-cancer (A) andare cancer site-specific.

FIGS. 3A-3J show western blots of 2-D gel electrophoresis/western blotsof sera from cancer patients analyzed individually. Cancer sites arepresented in the order of decreasing molecular weight of the majortranscript variant present. FIG. 3A. Cervical cancer. FIG. 3B. Ovariancancer. FIG. 3C. Prostate cancer. FIG. 3D. Breast cancer. FIG. 3E.Non-small cell lung cancer. FIG. 3F. Small cell lung cancer. FIG. 3G.Pancreatic cancer. FIG. 3H. Colon cancer. FIG. 3I. Non-Hodgkinslymphoma. FIG. 3J. Melanoma. The approximate location of unreactive (atbackground) albumin (Ab) is labeled for comparison. Approximately 180non-cancer patient sera were analyzed in parallel without evidence ofproteins indicative of specific transcript variants.

FIGS. 4A-4B show analytical gel electrophoresis and immunoblots ofpatient sera. FIG. 4A. Sera from a patient with non-small cell lungcancer contains a 54 kDa, isoelectric point pH 5.1 transcript variant(arrow). FIG. 4B. Sera from a patient with small cell lung cancercontains a 52 kDa, isoelectric point 4.3 transcript variant (arrow). Thereference spots to the right, Mr 52 kDa and isoelectric point pH 4.1 areα1-antitrypsin inhibitor. Albumin and other serum proteins areunreactive.

FIGS. 5A-5E show 2-D gel electrophoretic separations and detection ofENOX2 transcript variants specific for breast cancer by western blottingof patient sera in FIGS. 5A-5E. Arrow=66 to 68 kDa breast cancerspecific transcript variant. R=52 kDa, isoelectric point pH 4.1α1-antitrypsin inhibitor reference spot.

FIG. 6 is an interpretive diagram to illustrate the various stages ofcancer progression (estimated to require as long as 20 y) beginning witha cancer-causing event (initiation) through development of a clinicallydefined malignancy.

DETAILED DESCRIPTION OF THE INVENTION

The cancer diagnostic system of the present invention utilizestwo-dimensional polyacrylamide gel electrophoretic techniques for theseparation of proteins in human sera to generate cancer-specific isoformpatterns and compositions indicative of cancer presence, tumor type,disease severity and therapeutic response. The protocol is designed forthe detection of at least 20 cancer-specific ENOX2 isoforms which areresolved to indicate cancer presence and disease severity. Thisspecification illustrates the process of the isoform-resolvingtwo-dimensional gel electrophoresis protocol and subsequentimmunoanalysis to detect ENOX2 isoforms which reflect particularcancers.

Two-dimensional gel electrophoresis separates by displacement in twodimensions oriented at right angles to one another and immunoblottingidentifies the ENOX2 isoforms. In the first dimension isoforms areseparated according to charge (pI) by isoelectric focusing (IEF). Theisoforms are then separated according to size (Mr) by SDS-PAGE in asecond dimension. The isoforms are then blotted onto a nitrocellulosemembrane for further analysis using a pan-cancer specific antibodypreparation.

Ecto-Nicotinamide Adenine Dinucleotide Oxidase DisulfideThiol Exchanger2 (ENOX2) (GenBank accession no. AF207881; Chueh et al., 2002) alsoknown as Tumor Associated Nicotinamide Adenine Dinucleotide Oxidase(ENOX2) is ideally suited as a target for early diagnosis as well as forearly preventive intervention (FIG. 1). The proteins are expressed onthe cell surface of malignancies and detectable in the serum of patientswith cancer (Cho et al., 2002). ENOX2 proteins are terminal hydroquinoneoxidases of plasma membrane electron transport. From the standpoint ofearly intervention, they are important in the growth and enlargement oftumor cells (Morre and Morre, 2003; Tang et al., 2007. Biochemistry46:12337-12346; 2008. Oncol. Res. 16:557-567). Our approach using ENOX2,as a target for both early detection and for early interventions, isbased on these properties (Cho et al., 2002; Morre and Morre, 2003;reviewed by Davies and Bozzo, 2005. Drug News Perspect 19: 223-225).While ENXO2 presence provides a non-invasive approach to cancerdetection, without methodology to identify cancer site-specific ENOX2forms, it did not offer an indication as to cancer type or location.

The opportunity to simultaneously determine both cancer presence andcancer site emerged as a result of 2-dimensional gel electrophoreticseparations where western blots with a pan ENOX2 recombinant singlechain variable region (ScFv) antibody carrying an S tag (FIGS. 2A and2B) was employed for detection (Hostetler et al., 2009. Clin. Proteomics5: 46-51). The antibody cross reacted with all known ENOX2 forms fromhematological and solid tumors of human origin but, of itself, did notdifferentiate among different kinds of cancers. Analyses using thisantibody, when combined with two-dimensional gel electrophoreticseparation, revealed specific ENOX2 species subsequently identified astranscript variants, each with a characteristic molecular weight andisoelectric point indicative of a particular form of cancer (Hostetleret al, 2009; Table I).

ENOX transcript variants of specific molecular weights and isoelectricpoints are produced uniquely by patients with cancer. The proteins areshed into the circulation and have the potential to serve as definitive,non-invasive and sensitive serum markers for early detection of bothprimary and recurrent cancer in at risk populations with a low incidenceof false positives, as they are molecular signature molecules producedspecifically by cancer cells and absent from non-cancer cells.

As the 2-D-western blot protocol detects cancer early, well in advanceof clinical symptoms. The opportunity to combine early detection withearly intervention as a potentially curative prevention strategy forcancer by eliminating the disease in its very earliest stages is unique.

Analytical 2-D gel electrophoresis and immunoblotting of ENOX proteinsfrom a mixed population of cancer patients (cervical, breast, ovarian,lung and colon carcinomas, leukemias and lymphomas) revealed multiplespecies of acidic proteins of molecular weight between 34 and 100 kDa inquadrants I and IV (FIG. 1), none of which were present in sera ofnon-cancer patients (FIG. 1) (Hostetler et al., 2009). Separation in thefirst dimension was by isoelectric focusing over the pH range of 3 to 10and separation in the second dimension was by 10 percent SDS-PAGE.Isoelectric points of the ENOX2 transcript variants were in the range of3.9 to 6.3. The principal reactive proteins other than the ENOX2 formswere a 53 kDa isoelectric point pH 4.1, mostly phosphorylatedα1-antitrypsin inhibitor (α2-HS-glycoprotein; fetuin A) (Labeled “R” inFIGS. 2A and 2B) which served as a convenient loading control andisoelectric point reference and a 79-85 kDa, isoelectric point pH 6.8serotransferrin which served as a second point of reference for loadingand as an isoelectric point reference (Table II). The two cross reactivereference proteins are present in a majority of sera and plasma of bothcancer and non-cancer subjects. Albumin and other serum proteins do notreact. On some blots, the recombinant scFv was weakly cross-reactivewith heavy (ca. 52 kDa) and light (ca. 25 kDa) immunoglobulin chains.

Sera from individual patients with various forms of cancer were analyzedby 2-D gel electrophoresis and immunoblotting to assign each of theENOX2 isoforms of FIGS. 2A and 2B to a cancer of a particular tissue oforigin (Table 1). Sera of breast cancer patients contained only the 64to 68 kDa ENOX2 (FIG. 3D) and the α1-antitrypsin inhibitor referenceprotein (FIGS. 3A and 3J). Sera from cervical cancer patients containedthe 94 kDa ENOX2 transcript variant (FIG. 3A). Sera from ovarian cancerpatients contained ENOX1 transcript variants of 80 kDa and 40.5 b kDa(FIG. 3B). Sera from patients with prostate cancer contained one or more75 kDa ENOX2 transcript variants resulting in small variations inisoelectric points (FIG. 3C). Sera from patients with non-small celllung carcinoma contained a 52 kDa ENOX2 transcript variant while serafrom non-small cell lung carcinoma patients contained a of 52 kDa ENOX2transcript variant (FIGS. 3E and 3F and FIGS. 4A and 4B). ENOX2transcript variants of 50 and 52 kDa characterized sera of pancreaticcancer patients (FIG. 3G) whereas sera of colon cancer patientscontained ENOCX2 transcript variants of 52 kDa and 43 kDa (FIG. 3H).FIG. 3I from sera of a patient with non-Hodgkin's lymphoma illustratesthe 45 kDa ENOX2 transcript variant of low isoelectric pointcharacteristic of leukemias and lymphomas. Sera of patients withmalignant melanoma contained an ENOX2 transcript variant of 38 kDa (FIG.3J).

Particularly relevant are observations where the 64 to 68 kDa ENOX2transcript variant (pH 4.5) of sera correlated with disease presence inboth late (Stage IV) (FIG. 5A) and early (Stage I) (FIG. 5F) disease andin Stage IV recurrence (FIG. 5C) but was absent from sera of non-cancer(normal) volunteers (FIG. 5B) or in survivors free of disease for one tofive years (FIG. 5D). Additionally, the 64 to 68 kDa breastcancer-specific transcript variant does not apply to a subset of breastcancer patients but appears to be universally present. Analyses of seraof more than 60 patients with active disease including 20 Stage I andStage II breast cancer patients all tested positive.

Unlike most published cancer markers, cancer-specific ENOX2 variants arenot simply present as elevated levels of a serum constituent present inlesser amounts in the absence of cancer. The cancer-specific ENOX2transcript variants result from cancer-specific expression ofalternatively spliced mRNAs (Tang et al., 2007; 2008). Neither thesplice variant mRNAs nor the ENOX2 isoform proteins are present indetectable levels in non-cancer cells or in sera of subjects withoutcancer (Table I).

Findings from a separate study with small cell and non-small cell lungcancer suggest that the 2-D-western blot test detects cancer presence 5to 7 years in advance of the appearance of clinical symptoms. Thissupposition is based mainly on our analysis of two special cancer panelsof sera obtained through the Early Detection Research Network (EDRN) ofthe National Cancer Institute. One panel consisted of about 20 knownlung cancer patient sera and 35 control patient sera. Using the2-D-western blot protocol to identify specific ENOX2 isoforms, wesuccessfully identified all 20 of the known lung cancer patient sera.However, unexpectedly a high incidence of ENOX2 presence was encounteredin sera from the “control” group which were obtained from a communityscreening study. From additional information obtained through the EDRN,16 of the 17 positive control subject samples where our findingsspecifically indicated lung cancer (the lung cancer ENOX2 markers werefound) were smokers with smoking histories in the range of 15 to 88pack-years. However, the anticipated incidence of undetected lungcancers in such a population would be in the order of 10% or less ratherthan nearly 50%. Since the aberrant ENOX2 transcript variants associatedwith lung cancer, are single molecular species produced only by lungcancer, the possibility was raised that lung cancer was being detectedmuch earlier than was currently possible by other methods. Theindications might be as early as 5 to 7 years before clinical symptoms,based on the estimated 20 year development time for lung cancerexpression between carcinogen exposure and a clinically evident cancer(Petro et al., 2000. Br. Med. J. 231: 323-329) as diagrammed in FIG. 6.

Similar results were obtained with a panel of female subjects at riskfor breast and ovarian cancer. An analysis of a panel of 127 sera in aBiomarker Reference Set for Cancers in Women also provided through theEarly Detection Research Network of the National Cancer Institutesupport our indications that the 2-D gel-western blot system is able todetect cancer presence 5 to 7 years in advance of clinical symptoms. Thepanel consisted of samples pooled form 441 women in 12 differentgynecologic and breast disease categories plus 115 sera from age-matchedcontrol women. Of the 127 sera samples in the panel 29 tested positivefor breast cancer and another 16 tested positive for ovarian cancer.Since the aberrant transcript variants are single molecular speciesproduced by specific cancers such as lung, breast or ovarian, thefindings suggest that cancer was being detected in the controlpopulation much earlier than is currently possible by other methods. Asestimated for lung cancer, the indications might be as early as 5 to 7years before clinical symptoms based on the estimated development timeestimated for breast as well as lung cancer expression between a cancercausing event and clinically evident disease (Weinberg, 2007. TheBiology of Cancer, Garland Science).

Many cancers are detected only after clinical symptoms present and oftenafter the cancer has spread leaving chemotherapy as perhaps the onlyresource for treatment. Tomographic or x-ray methods may detect beforeclinical symptoms present but only after a tumor mass has alreadyformed. There appear to be few, if any, on-going indications ofopportunities either for early cancer detection or for earlyintervention. Various genomic, transcriptomic and/or proteomic analyses,while of potential utility for tissue analyses of biopsy material, havethus far failed to provide new and reliable non-invasive serumindicators of cancer occurrence (Goncalves and Bertucci, 2011. Med.Prin. Pract. 204: 4-18) despite continued promise offered by circulatingmicroRNAs (Wu et al., 2011. J. Biomed. Biotechnol. Article ID 597145). Arelatively small percentage of all cancers can be attributed topredisposing genes such as BRACA1, BRACA2 and less frequently p53 andPTEN (Lee et al., 2010. Breast Cancer Res. Treat. 122: 11-25) for 5 to10% of all breast cancers. While indicative of cancer risk, predisposinggenes do not necessarily signal cancer presence.

Table I shows that sera from patients with different cancers exhibitdistinct patterns of ENOX2 isoforms with characteristic molecular weightand isoelectric points (pH). Updated from Hostetler et al. (2009).

Sera Molecular Isoelectric Cancer analyzed weight point, pH Cervical 1894 kDa 5.4 Ovarian 41 80 and 40.5 kDa   4.2 and 4.1 Prostate 70 75 kDa6.3 Breast/Uterine 55  64 to 68 kDa 4.5 Non-small cell lung 83 54 kDa5.1 Small cell lung 22 52 kDa 4.3 Pancreatic 24 50 kDa 4.3 Colon 55 52and 34 kDa 4.3 and 3.9 Lymphoma, Leukemia 16 45 kDa 3.9 Melanoma 12 38kDa 5.1

Table II provides protein sequence similarity between ENOX2 and the tworeference proteins α1-anti-trypsin inhibitor and serrotransferrinreactive with the pan ENOX2 scFv recombinant antibody. Regions ofsimilarity are restricted to a 7 amino acid sequence (underlined)adjacent in ENOX2 to the EEMTE SEQ ID NO:20 quinone inhibitor-bindingsite which serves as the antigen sequence to which the specific scFvantibodies bind.

ENOX2 (SEQ ID NO: 17) EEMTETKETEESALVS α-antitrypsin inhibitor(SEQ ID NO: 18) GTDCVAKEATEAAKCN Serrotransferrin (SEQ ID NO: 19)CLDGTRKPVEEYANCHDetails of MethodA. Sample Preparation

1. Prepare/thaw re-hydration solution (−20, 1.5 mL tube labeled RB)

a. Add 1% Dithiothreitol (DTT) to solution before use (0.01 g/1.0 mL)

2. Add 120 μL of Rehydration Buffer to a 1.7 ml tube

3. Add 30 μL of sera to tube

4. Vortex solution until fully mixed

5. Remove Immobiline DryStrips from freezer (−20° C., pH 3-10) and allowstrips to equilibrate to RT for 5 minutes.

a. Do not leave strips at RT for more than 10 min.

6. Record ID# from strip

7. Load 130 μL of sample to tray per 7 cm DryStrip. Ensure tray islevel.

8. Place DryStrips gel-side down over sample

9. Ensure sample is evenly spread throughout strip by carefully liftingstrip in and out of sample a few times if needed.

10. If samples are concentrated in one region of the strip, redistributesample by pipetting.

11. Remove air bubbles by gently pressing down on Dry Strip with pipettetip.

12. Place lid on tray and place tray in plastic bag with ddi-H₂O soakedpaper towels.

13. Seal bag.

14. Allow sample to re-hydrate overnight at RT on a level surfaceallowing strips to absorb sample for 12-24 hrs.

B. Isoelectric Focusing (First Dimension)

1. Turn on IPGphor (ensure proper startup of machine)

2. Place strips on Manifold focusing tray as follows

a. Gel side up

b. Positive (acidic) end towards back

c. Strips are aligned

d. Between metal strips (so electrodes fit and touch metal strip)

3. Obtain 2 Paper Wicks per strip

4. Wet wicks with 150 μL ddi-H₂O per wick.

5. Place wicks over anodic and cathodic ends of gel (approx. 0.3 cm).

6. Place electrodes on wicks, but away from gel (be sure prong is onmetal plate), and lock in place.

7. Cover strips with DryStrip Cover fluid

a. Fill strips entire lane with oil

b. Ensure strips are fully covered

8. Close lid

9. IEF run with IPGphor II

a. Maximum amperage: 50 μAmps

b. Temperature: 20° C.

c. Ensure correct assembly by checking initial voltage

d. As needed, pause run and replace wicks, continue run until dye frontdisappears.

Step Voltage Time/Vhrs 7 cm Strip pH 3-10 1 - Stp. 250 V 250 Vhrs. (Run#1) 2 - Stp. 500 V 500 Vhrs. 3 - Stp. 1000 V 1000 Vhrs. 4 - Grd. 4000 V3 hrs. 5 - Stp. 4000 V 25,000 Vhrs. 6 - Stp. 500 V HoldC. Prepare SDS-Page Gels (for Second Dimension)

1. Prior to use, wash and scrub plates very well in soap and hot water.

2. Rinse in diH₂O.

3. Leave the plates to air dry or wipe with ethanol-soaked Kimwipes.

4. Order plates in Protean-plus Multi-Gel casting Chamber (Bio-Rad) asper manual (with a spacer between each plate and block).

5. Ensure screws are fully tightened.

6. Add gel solution

7. Stop pouring when gel is about 1-1.5 cm from top of glass plates.

8. Gently overlay gels with ethanol

9. Cover with Saran Wrap.

10. Allow gels to polymerize for at least one hour (best if overnight)

D. Equilibration (First Dimension)

1. Remove strips from tray and place on Kimwipe to remove excess oil.

1. Place strips gel side up on Kimwipe

2. Overlay strips with a second Kimwipe and gently blot to remove oil.

2. Place strips in equilibration plate gel side up; freeze orequilibrate.

1. Freeze: Wrap plate in plastic wrap, store at −80° C.

i. Thaw strips prior to equilibration (clear when thawed).

2. Equilibrate: continue to next step

3. Cover strips with equilibration buffer, about 1.5 mL per strip.

4. Heat up Agarose until it is liquefied

5. Shake 20 min at RT

E. SDS-PAGE (Second Dimension)

1. Prepare left (pH 10 side) markers by adding 8 μL of standards onWhatman 3 MM chromatography paper cut to about 3 cm.times.0.75 cm.

1. Standards should be added to bottom of paper, about 1 cm high.

2. Prepare right (pH 3 side) markers by adding 8 μL of standards onWhatman 3 MM chromatography paper cut to approximately 3 cm.times.0.75

1. Standards should be added to bottom of paper, about 1 cm high

3. Pour off Equilibration Buffer.

4. Cover strips in SDS Running buffer to rinse away excess EquilibrationBuffer.

5. Remove SDS Running buffer from strips.

6. Repeat SDS Running buffer rinse.

7. Carefully place strips gel side out on back plate of SDS-PAGE gel.

8. Overlay strips with 1% low melting agarose once it has cooled enoughto touch skin

1. Ensure no air bubbles have formed under the gel.

2. Use ruler to tap gel and remove air bubbles.

9. Insert marker's next to appropriate end of IEF strip, ensuring markeris flush to the gel on the strip

10. Allow polymerization of agarose

11. Continue for each strip to be loaded in 2.sup.nd dimension

12. Place gels in Dodeca tank, HINGED SIDE DOWN

13. After all gels have been put in tank, ensure gels are covered inentirety by SDS running buffer

14. 2.sup.nd dimension run is done at 13° C.

1. 250 V

2. 1-1.5 hr. (allow gel to run until gel front approaches tubing in lidof tank).

F. Protein Transfer (for Western Blot)

1. Remove gel from Dodeca tank

2. Cut gel to desired size

3. Fill tray (large enough to fit gel) with transfer buffer.

4. Place sponges in transblot cell—2 sponges per gel

5. Fill tank with transfer buffer to allow sponges to saturate withtransfer buffer

6. Soak pre-cut transfer membrane

7. Assemble transfer cassette as follows

1. Black side down

2. Sponge soaked in transfer buffer

3. Filter paper

4. Gel

5. Nitrocellulose membrane—once placed on gel do not move membrane

6. Filter Paper

7. Sponge

8. Ensure all air bubbles have been removed between gel and membrane

9. Place tray in Transblot tank, black side (gel side) of tray to blacktank side

10. Transfer at 4° C. and following conditions (transfer can be done inan ice bath if needed)

1. 90 V for 50 min.

2. Membrane can be left in tank overnight at 4° C. after transfer.

G. Immunological Analysis for Western Blot Using ScFv with S-Tag Linkedto Alkaline Phosphatase as Antibody

1. Remove membrane from transfer

2. Rinse membrane in 1% milk (enough to cover membrane) and block, 10min, RT

3. Prepare antibody solution (According to Titer instructions on Ab)

4. Remove blocking solution (save at 4° C.)

5. Place membrane into container with antibody solution

6. Incubate at 4° C. overnight (usually 8-12 hr)

H. Development of Western Blot and Scanning

1. Remove 1° antibody

2. Wash membrane 4×

1. Cover membrane with TBST

2. Gently shake at RT for 5 min.

3. Cover membrane with Western Blue

4. Allow to develop until reference spots reach maximum intensity

5. Stop develop by rinsing with ddi-water

6. Dry membrane

7. Scan membrane

Solutions Used for First Dimension

I. Rehydration Buffer pH 7 (25 mL):

Amount Molar Mass Final Chemical Added (g/mol) Value Urea 10.51 g 60.067M Thiourea 3.81 g 76.12 2M CHAPS 0.5 g 614.88  2% asb-14 0.125 g 434.680.5% 40% Ampholytes 330 μL N/A 0.5% IPG Buffer 125 μL N/A 0.5% ddi-H₂OTo 25 mL 18.002 N/A Bromophenol Blue 3 mg 669.96 0.012%  Dissolve Ureain minimal ddi-H2O(do not heat over 30° C.). Dissolve Thiourea inUrea/ddi-H₂O solution, and then add remaining chemicals. Q.S. with to 25mL and aliquot to 1 mL tubes and store at −80° C. Add 1% DTT - 10 mg(0.01 g) before use.Solutions Used for Second DimensionTris Buffer (1.5 M, pH 8.8) (1 L):

Amount Molar Mass Final Chemical Added (g/mol) Value Trizma 181.65 g121.14 1.5M HCl pH to 8.8 36.46 N/A ddi-H₂O To 1 L 18.02 N/A DissolveTrizma in 750 mL and adjust pH to 8.8 with HCl. Q.S. to final volume of1 L with ddi-H₂O and store at 4° C.Equilibration Buffer (400 mL):

Amount Molar Mass Final Chemical Added (g/mol) Value Tris-Buffer 134 mLN/A 0.5M   (1.5M, pH 8.8) Urea 144.14 g 60.06 6M Glycerol 120 mL (150 g)92.09 30% SDS 10 g 288.38 2.5%  ddi-H₂O To 400 ml 18.02 N/A BromophenolBlue Trace amount 669.96 N/A Q.S. to final volume of 4 L with ddi-H₂O.Add Bromophenol Blue (add with pipette tip to give trace of blue).Aliquot to 15 mL tubes and store at −20° C.Acrylamide Gel (20.times.20; 1-mm Thick)

Tris Buffer 1.5M, 30% Gel # pH 8.8 Acrylamide ddi-H₂O 10% APS TEMED %gels (mL) (mL) (mL) (mL) (mL) 10 6 100 133.33 166.67 4 0.4 8 125 166.67208.33 5 0.5 10 150 200 250 6 0.6 12 175 233.33 291.67 7 0.7Formulations for Protean II gels10% APS

Amount Molar Mass Final Chemical Added (g/mol) Value APS 2 g 228.18 10%ddi-H₂O To 20 mL 18.02 N/A Q.S. to final volume of 20 mL with ddi-H₂O.Agarose Solution (1%):

Amount Molar Mass Final Chemical Added (g/mol) Value Agarose 1.5 g N/A1% SDS-Running Buffer 150 mL N/A N/A Bromophenol Blue Trace amount669.96 N/A Combine agarose and SDS-Run Buffer. Microwave to heat anddissolve. Add Bromophenol Blue (add with pipette tip to give trace ofblue).10×Running Buffer (4 L):

Amount Molar Mass Final Chemical Added (g/mol) Value Trizma 121.2 g121.14 0.25M Glycine 576 g 75.07 1.92M SDS 40 g 288.38 1% ddi-H₂O To 4 L18.02 N/A Q.S. to final volume of 4 L with ddi-H₂O.Solutions for Western BlotWestern Transfer Buffer (4 L):

Amount Molar Mass Final Chemical Added (g/mol) Value Trizma 121.2 g121.14 0.025M Glycine 57.6 g 75.07 0.192M Methanol 480 mL 32.04 0.12%SDS 3 g 288.38 .075% ddi-H₂O To 4 L 18.02 N/A Q.S. to final volume of 4L with di-H₂O.Blocking Buffer (5% BSA)

Molar Mass Final Chemical Mass (g/mol) Value BSA 5 g 66500  5% N₃Na 0.2g 65.01 0.2% TBST To 100 mL 18.02 N/A Q.S. to final volume of 100 mLwith TBST.10×TBST (4 L)

Amount Molar Mass Final Chemical Added (g/mol) Value Trizma 48.4 g121.14 100 mM NaCl 350.6 g 58.44 1.5M Tween 20 20.6 g 1227.54 0.5% HClpH to 7.5 36.46 N/A ddi-H₂O To 4 L 18.02 N/A Add Trizma and NaCl to 3.5L ddi-H₂O. pH to 7.5 by HCl. Add Tween 20. Q.S. to 4 L with ddi-H₂O.Summary of 2-D Gel Electrophoresis Western Blot Early Detection Protocol

Serum was prepared from 5 ml of blood collected by venipuncture (withtourniquet) in standard B & D 13.times.100 (7 ml) vacutainer clot tubes(or equivalent) with or without hemoguard closure. After approximately30 min at room temperature to allow for clotting, the clot was pelletedby centrifugation for 5 to 10 min at 2,500 to 3,000 rpm. Clot-free serumwas decanted into a clean tube, labeled and analyzed fresh or storedfrozen.

For western blot analysis, 30 μl of sera was added to 120 μl ofRehydration Buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS[(3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), anondenaturing zwitterionic detergent], 0.5% (w/v) ASB-14(amidosulfobetaine-14, a zwitterionic detergent), 0.5% (v/v) ampholytespH 3-10 (Bio-Rad), 0.5% (v/v) immobilized pH gradient (IPG) buffer pH3-10 (Amersham-Pharmacia Biotech) and 65 mM dithiothreitol). The sampleswere quickly vortexed to mix sera with Rehydration Buffer. Four to sixmg of protein were loaded for analysis. The samples were electrophoresedin the first dimension by using a commercial flatbed electrophoresissystem (Ettan IPGphor 3, Amersham-Pharmacia Biotech) with IPG dry strips(Amersham). A linear pH range of 3 to 10 on 7 cm IPG strips was used.The IPG strips were rehydrated with the samples overnight at roomtemperature. The strips were then focused at 50 mA per strip and atincreasing voltage of 250 V for 250 Vhrs, 500 V for 500 Vhrs, 1,000 Vfor 1,000 Vhrs and 4,000 V for 3 hrs. The samples were then focused at aconstant 4,000 V for 28,000 Volt-hours. After isoelectric focusing, theIPG strips were re-equilibrated for 20 min in 2.5% (w/v) SDS, 6 M urea,30% (v/v) glycerol, 100 mM Tris-HCl (pH 8.8). The strips were placedonto linear SDS-PAGE gels (10% (w/v) polyacrylamide) and electrophoresedat a constant 250 V for 75 min. The samples were then transferred tonitrocellulose membranes by electroblotting using the Bio-Rad Trans-BlotElectrophoretic Transfer Cell. The membranes were blocked using milkprotein (1% low fat dry milk) at room temperature for 10 min. Detectionwas with recombinant anti-ENOX2 single chain variable region of antibody(scFv) that was alkaline phosphatase-linked overnight at 4° C. Afterwashing, detection was performed with Western Blue nitrotetrazolium(NBT) substrate (Promega, Madison, Wis.; Cat. No. 53841) at roomtemperature. Images were scanned and processed using Adobe Photoshop.Quantitation utilized an algorithm developed for this purpose. Reactiveproteins appeared reddish blue. For interpretative purposes, the blotswere divided into quadrants I-IV with unreactive serum albumin at thecenter (FIGS. 2A and 2B).

EXAMPLES Example 1 Analysis of Sera Pooled from Cancer Patients

Example 1A. NOX-enriched serum proteins (approximately 4-6 mg) from serapooled from cancer patients (breast, ovarian, lung and colon) wereresolved by 2-D gel electrophoresis. Detection was by recombinantanti-ECTO-NOX antibody (single chain variable region ScFv) carrying anS-tag followed by alkaline phosphatase-linked anti-S with Western BlueNBT alkaline phosphatase substrate yield several proteins present in thecancer sera (FIG. 1) but absent from sera of non-cancer patients orhealthy volunteers. Example 1B. The same procedure can be followed as inExample 1A, except that detection can be by recombinant anti-ECTO-NOXvariable region single chain (scFv) using the scFv linked directly asdescribed above to alkaline phosphatase (overnight at 4° C.). By usingthe directly linked antibody the process is less expensive and one dayfaster than using an S-tag followed by an anti-S tag antibody linked toalkaline phosphatase.

Example 2 Analysis of Small Cell Lung Cancer Patient Serum

Example 2A. 2-D gel analysis when applied to sera of a patient withsmall cell lung cancer contained a 52 kDa, pH 4.2 ENOX2 protein inquadrant I with detection using the S-tag procedure in Example 1A above.Example 2B. The same procedure can be followed as in Example 2A, exceptthat detection can be by recombinant anti-ECTO-NOX variable regionsingle chain (scFv) using the scFv linked directly as described above toalkaline phosphatase (overnight at 4° C.). By using the directly linkedantibody the process is less expensive and one day faster than using anS-tag followed by an anti-S tag antibody linked to alkaline phosphatase.

Example 3 Analysis of Non Small Cell Lung Cancer Patient Serum

Example 3A. 2-D gel analysis as in FIG. 1 when applied to plasma from apatient with non-small cell lung cancer revealed a non-small cell cancerspecific ENOX2 isoform at 54 kDa, pH 5.1 in quadrant 1 with detectionusing the S-tag procedure in Example 1A above. Example 3B. The sameprocedure can be followed as in Example 3A, except that detection can beby recombinant anti-ECTO-NOX variable region single chain (scFv) usingthe scFv linked directly as described above to alkaline phosphatase(overnight at 4° C.). By using the directly linked antibody the processis less expensive and one day faster than using an S-tag followed by ananti-S tag antibody linked to alkaline phosphatase.

Example 4 Analysis of Breast Cancer Patient Serum

Example 4A. 2-D gel analysis as in FIG. 1 when applied to sera from apatient with breast cancer revealed a breast cancer-specific ENOX2protein of 68 kDa, pH 4.5 in quadrant 1 with detection using the S-tagprocedure in Example 1A above. Example 4B. The same procedure can befollowed as in Example 4A, except that detection can be by recombinantanti-ECTO-NOX variable region single chain (scFv) using the scFv linkeddirectly as described above to alkaline phosphatase (overnight at 4°C.). By using the directly linked antibody the process is less expensiveand one day faster than using an S-tag followed by an anti-S tagantibody linked to alkaline phosphatase.

Example 5 Analysis of Prostate Cancer Patient Serum

Example 5A. 2-D gel analysis as in FIG. 1 when applied to sera from apatient with prostate cancer revealed prostate cancer-specific ENOX2isoforms at 75 kDa and isoelectric points of pH 6.3 with detection usingthe S-tag procedure in Example 1A above. Example 5B. The same procedurecan be followed as in Example 5A, except that detection can be byrecombinant anti-ECTO-NOX variable region single chain (scFv) using thescFv linked directly as described above to alkaline phosphatase(overnight at 4° C.). By using the directly linked antibody the processis less expensive and one day faster than using an S-tag followed by ananti-S tag antibody linked to alkaline phosphatase.

Example 6 Analysis of Cervical Cancer Patient Serum

Example 6A. 2-D gel analysis as in FIG. 1 when applied to serum from apatient with cervical cancer revealed a cervical cancer-specific ENOX2isoform at 94 kDa, pH 5.4 with detection using the S-tag procedure inExample 1A above. Example 6B. The same procedure can be followed as inExample 6A, except that detection can be by recombinant anti-ECTO-NOXvariable region single chain (scFv) using the scFv linked directly asdescribed above to alkaline phosphatase (overnight at 4° C.). By usingthe directly linked antibody the process is less expensive and one dayfaster than using an S-tag followed by an anti-S tag antibody linked toalkaline phosphatase.

Example 7 Analysis of Colon Cancer Patient Serum

Example 7A. 2-D gel analysis as in FIG. 1 when applied to serum from apatient with colon cancer revealed colon cancer-specific ENOX2 isoformsat 38 and 52 kDa, pH 4.3 and 3.9 with detection using the S-tagprocedure in Example 1A above. Example 7B. The same procedure can befollowed as in Example 7A, except that detection can be by recombinantanti-ECTO-NOX variable region single chain (scFv) using the scFv linkeddirectly as described above to alkaline phosphatase (overnight at 4°C.). By using the directly linked antibody the process is less expensiveand one day faster than using an S-tag followed by an anti-S tagantibody linked to alkaline phosphatase.

Example 8 Cancer Specific Isoforms

For each kind of cancer there appears to be a ENOX2 isoform (ovarian,breast, cervical, colon, non-small cell lung, prostate small cell lung)or combination of ENOX2 isoforms that is specific to the tissue or celltype of origin for the cancer. This test is preferably done withrecombinant anti-ECTO-NOX variable region single chain (scFv) using thescFv linked directly as described above to alkaline phosphatase. Byusing the directly linked antibody the process is less expensive and oneday faster than using an S-tag followed by an anti-S tag antibody linkedto alkaline phosphatase.

Example 9 Analysis of Patient Serum where Cancer of Unknown Origin

The 2-D gel of sera from a patient with cancer where the primary tumorwas unknown revealed the presence of 40.5 and 80 kDa, pH 4.2 and 4.1ENOX2 isoforms to indicate that the primary cancer was ovarian cancer(not shown).

In more than 25 randomly selected outpatient sera and sera of healthyvolunteers, both quadrants I and IV of the 2-D gels were devoid of ENOX2isoforms, confirming previous observations that ENOX2 proteins areabsent from non-cancer patients or sera of healthy volunteers.

The diagnostic strategy of the invention combines one- andtwo-dimensional polyacrylamide gel electrophoretic separations of humansera to generate cancer specific isoform patterns and compositionsindicative of cancer presence, tumor type, disease severity andtherapeutic response. At least 20 cancer-specific ENOX2 isoforms areresolved indicative of cancer presence and disease severity. Detectionuses a recombinant single chain antibody (scFv) that reacts with allknown ECTO-NOX isoforms of human origin. While the technique can be usedwith an antibody that has an S-tag, the process is less expensive andfaster by using the scFv linked directly to alkaline phosphatase oranother suitable detection aid.

Monoclonal antibody generated against ENOX2 NADH oxidase tumor cellspecific was produced in sp-2 myeloma cells; however, the monoclonalantibody slowed the growth of sp-2 myeloma cells that were used forfusion with spleen cells after 72 h. This phenomenon made it difficultto produce antibody in quantity. To overcome this problem, the codingsequences of the antigen-binding variable region of the heavy chain andthe light chain (Fv region) of the antibody cDNA were cloned and linkedinto one chimeric gene, upstream of the S-tag coding sequence. The Fvportion of an antibody, consisting of variable heavy (VH) and variablelight (VL) domains, can maintain the binding specificity and affinity ofthe original antibody (Glockshuber et al. 1990. Biochemistry29:1262-1367).

For a recombinant antibody, cDNAs encoding the variable regions ofimmunoglobulin heavy chain (VH) and light chain (VI), are cloned byusing degenerative primers. Mammalian immunoglobulins of light and heavychain contain conserved regions adjacent to the hypervariablecomplementary defining regions (CDRs). Degenerate oligoprimer sets allowthese regions to be amplified using PCR (Jones et al. 1991.Bio/Technology 9:88-89; Daugherty et al. 1991. Nucleic Acids Research19:2471-2476). Recombinant DNA techniques have facilitated thestabilization of variable fragments by covalently linking the twofragments by a polypeptide linker (Huston et al. 1988. Proc. Natl. Acad.Sci. USA 85:5879-5883). Either VL or VH can provide the NH2-terminaldomain of the single chain variable fragment (ScFv). The linker shouldbe designed to resist proteolysis and to minimize protein aggregation.Linker length and sequences contribute and control flexibility andinteraction with ScFv and antigen. The most widely used linkers havesequences consisting of glycine (Gly) and serine (Ser) residues forflexibility, with charged residues as glutamic acid (Glu) and lysine(Lys) for solubility (Bird et al. 1988. Science 242:423-426; Huston etal. 1988. supra).

Total RNA was isolated from the hybridoma cells producing ENOX2-specificmonoclonal antibodies by the following procedure modified fromChomczynski et al. (1987) Anal. Biochem. 162:156-159 and Gough (1988)Anal. Biochem 176:93-95. Cells were harvested from medium and pelletedby centrifugation at 450.times.g for 10 min. Pellets were gentlyresuspended with 10 volumes of ice cold PBS and centrifuged again. Thesupernatant was discarded and cells were resuspended with an equalvolume of PBS. Denaturing solution (0.36 ml of 2-mercaptoethanol/50 mlof guanidinium stock solution-4M guanidinium thiocyanate, 25 mM sodiumcitrate, pH 7.0, 0.5% sarkosyl) 10 ml per 1 g of cell pellet was addedprior to use and mixed gently. Sodium acetate (pH 4.0, 1 ml of 2M), 10ml of phenol saturated water and 2 ml of chloroform:isoamyl alcohol(24:1) mixtures were sequentially added after each addition. Thesolution was mixed thoroughly by inversion. The solution was vigorouslyshaken for 10 sec, chilled on ice for 15 min and then centrifuged12,000.times.g for 30 min. The supernatant was transferred and an equalvolume of 2-propanol was added and placed at −20° C. overnight toprecipitate the RNA. The RNA was pelleted for 15 min at 12,000.times.g,and the pellet was resuspended with 2-3 ml of denaturing solution and 2volumes of ethanol. The solution was placed at −20° C. for 2 h, and thencentrifuged at 12,000.times.g for 15 min. The RNA pellet was washed with70% ethanol and then 100% ethanol. The pellet was resuspended withRNase-free water (DEPC-treated water) after centrifugation at 12,000×gfor 5 min. The amount of isolated RNA was measuredspectrophotometrically and calculated from the absorbance at 280 nm and260 nm.

The poly(A)mRNA isolation kit was purchased from Stratagene. Total RNAwas applied to an oligo(dT) cellulose column after heating the total RNAat 65° C. for 5 min. Before applying, the RNA samples were mixed with500 μl of 10× sample buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 MNaCl). The RNA samples were pushed through the column at a rate of 1drop every 2 sec. The eluates were pooled and reapplied to the columnand purified again. Preheated elution buffer (65° C.) was applied, andmRNA was eluted and collected in 1.5 ml of centrifuge tubes on ice. Theamount of mRNA was determined at OD260 (1 OD unit=40 μg of RNA). Theamounts of total RNA and mRNA obtained from 4.times.10.sup.8 cells were1328 μg and 28 μg, respectively.

mRNA (1-2 μg) dissolved in DEPC-treated water was used for cDNAsynthesis. mRNA isolated on three different dates was pooled forfirst-strand cDNA synthesis. The cDNA synthesis kit was purchased fromPharmacia Biotech. mRNA (1.5 μg/5 μl of DEPC-treated water) was heatedat 65° C. for 10 min. and cooled immediately on ice. The primed firststrand mix containing MuLV reverse transcriptase (11 μl) and appropriatebuffers for the reaction were mixed with mRNA sample. DTT solution (1 μlof 0.1 M) and RNase-free water (16 μl) also were added to the solution.The mixture was incubated for 1 h at 37° C.

Degenerate primers for light chain and heavy chain (Novagen, Madison,Wis.) were used for PCR. PCR synthesis was carried out in 100 μlreaction volumes in 0.5 ml microcentrifuge tubes by using Robocycler(Stratagene, La Jolla, Calif.). All PCR syntheses included 2 μl of senseand anti-sense primers (20 pmoles/μl), 1 μl of first-strand cDNA as atemplate, 2 μl of 10 mM of dNTPs, 1 μl of Vent polymerase (2 units/μl),10 μl of 10×PCR buffer (100 mM Tris-HCl, pH 8.8 at 25° C., 500 mM KCl,15 mM MgCl2, 1% Triton X-100), 82 μl of H2O. Triton X-100 ist-octylphenoxypolyethoxyethanol. All PCR profiles consisted of 1 min ofdenaturation at 94° C., 1 min of annealing at 55° C., and 1 min ofextension at 72° C. This sequence was repeated 30 times with a 6-minextension at 72° C. in the final cycle. PCR products were purified withQIAEX II gel extraction kit from Qiagen, Valencia, Calif. PCRamplification products for heavy and light chain coding sequences wereanalyzed by agarose gel electrophoresis and were about 340 base pair(bp) long and 325 bp long, respectively.

Total RNA or DNA was analyzed by agarose gel electrophoresis (1% agarosegels). Agarose (0.5 g in 50 ml of TAE buffer, 40 mM Tris-acetate, 1 mMEDTA) was heated for 2 min in a microwave to melt and evenly dispersethe agarose. The solution was cooled at room temperature, and ethidiumbromide (0.5 μg/ml) was added and poured into the apparatus. Each samplewas mixed with 6× gel loading buffer (0.25% bromophenol blue, 0.25%xylene cyanol FF, 40% (w/v) sucrose in water). TAE buffer was used asthe running buffer. Voltage (10 v/cm) was applied for 60-90 min.

According to the proper size for heavy and light chain cDNAs, the bandswere excised from the gels under UV illumination, and excised gels wereplaced in 1 ml syringes fitted with 18-gauge needles. Gels were crushedto a 1.5 ml Eppendorf tube. The barrel of each syringe was washed with200 μl of buffer-saturated phenol (pH 7.9.+−.0.2). The mixture wasthoroughly centrifuged and frozen at −70° C. for 10 min. The mixture wascentrifuged for 5 min, and the top aqueous phase was transferred to anew tube. The aqueous phase was extracted again with phenol/chloroform(1:1). After centrifuging for 5 min, the top aqueous phase wastransferred to a clean tube, and chloroform extraction was performed.Sodium acetate (10 volumes of 3 M) and 2.5 volumes of ice-cold ethanolwere added to the top aqueous phase to precipitate DNA at −20° C.overnight.

Purified heavy and light chain cDNAs were ligated into plasmid pSTBlue-1vector and transfected into NovaBlue competent cells (Stratagene).Colonies containing heavy and light chain DNAs were screened by blue andwhite colony selection and confirmed by PCR analysis. Heavy and lightchain DNAs were isolated and sequenced using standard techniques. Tables2A and 2B show the DNA sequences of heavy and light chain DNAs of ScFv.See also SEQ ID NO: 3 and SEQ ID NO: 4.

PCR amplification and the assembly of single ScFv gene was according toDavis et al. (1991) Bio/Technology 9:165-169. Plasmid pSTBlue-1 carryingVH and VL genes were combined with all four oligonucleotide primers in asingle PCR synthesis. Following first PCR synthesis, one tenth of thefirst PCR product was removed and added to a second PCR reaction mixturecontaining only the primer a (VH sense primer) and primer d (VLAntisense primer). The product of the second PCR synthesis yieldedsingle ScFv gene. The single ScFv gene was ligated to plasmid pT-Adv(Clontech, Palo Alto, Calif.). pT-Adv carrying ScFv gene was used forDNA sequencing.

The complete ScFv gene was assembled from the VH, VL and linker genes toyield a single ScFv gene by PCR (Tables 2A and 2B). The DNA sequenceencoding the linker was nucleotides long(GGAGGCGGTGGATCGGGCGGTGGCGGCTCGGGTGGCGGCGGCTCT; SEQ ID NO:6), whichtranslates to a peptide of 15 amino acids(GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer; SEQ ID NO:5). Primersfor PCR amplification are shown in Tables 2A and 2B. S-peptide waslinked to the C-terminus of ScFv[ScFv(S)]. S-peptide binds to S-proteinconjugated to alkaline phosphatase for Western blot analysis. The DNAsequence of the S-peptide isAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGC (SEQ ID NO:7) whichtranslates to S-peptide (LysGluThrAlaAlaAlaLysPheGluArgGln HisMetAspSer;SEQ ID NO:8).

Recombinant ScFv(S) was expressed in E. coli. First, oligo nucleotidesencoding S-peptide were linked to the 3′ end of the open reading frame(ORF) of ScFv DNA by PCR amplification. Incorporation of S-peptideenables to detect expressed ScFv protein by S-protein conjugated toalkaline phosphatase. The ENOX2-specific ScFv(S) coding sequence wasthen subcloned to plasmid pET-11a, a plasmid designed for proteinexpression in E. coli (Stratagene, Calif.). For PCR amplification, twoprimers were designed to amplify ORF of ScFv(S) containing endonucleaserestriction sites (NdeI and NheI) and S-peptide residues.

Plasmid pET-11a and ORF of ScFv(S) were digested with restrictionenzymes NdeI and NheI and ligated to produce plasmid pET11-ScFv(S). E.coli BL21 (DE3) was transformed with pET11-ScFv(S) and grown at 37° C.for 12 h in LB medium containing ampicillin (100 μg/ml). ScFv wasexpressed by addition of 0.5 mM IPTG and incubation for 4 h. Cells wereharvested and lysed using a French Pressure Cell (French Pressure CellPress, SLM Instruments, Inc.) (three passages at 20,000 psi). Cellextracts were centrifuged at 10,000×g for 20 min. Pellets containingdenatured inclusion bodies of ScFv were collected. Renaturation of theinclusion bodies of the ScFv was according to Goldberg et al. (1995)Folding & Design 1:21-27.

TABLE 1A DNA Sequence of Heavy chain ScFv (V_(H)), SEQ. ID NO: 1   1gaggtcaagc tgcaggagtc aggaactgaa gtggtaaagc ctggggcttc  51agtgaagttg tcctgcaagg cttctggcta catcttcaca agttatgata 101tagactgggt gaggcagacg cctgaacagg gacttgagtg gattggatgg 151atttttcctg gagaggggag tactgaatac aatgagaagt tcaagggcag 201ggccacactg agtgtagaca agtcctccag cacagcctat atggagctca 251ctaggctgac atctgaggac tctgctgtct atttctgtgc tagaggggac 301tactataggc gctactttga cttgtggggc caagggacca cggtcaccgt 351 ctcctca

TABLE 1B DNA Sequence of Light Chain ScR, (V_(L)), SEQ. ID NO: 2   1gaaaatgtgc tcacccagtc tccagcaatc atgtctgcat ctccagggga  51gagggtcacc atgacctgca gtgccagctc aagtatacgt tacatatatt 101ggtaccaaca gaagcctgga tcctccccca gactcctgat ttatgacaca 151tccaacgtgg ctcctggagt cccttttcgc ttcagtggca gtgggtctgg 201gacctcttat tctctcacaa tcaaccgaat ggaggctgag gatgctgcca 251cttattactg ccaggagtgg agtggttatc cgtacacgtt cggagggggg 301accaagctgg agctgaaagc g

TABLE 2A DNA Sequence for ScFv, SEQ. ID NO: 3   1 gtggtaaagc ctggggcttc 51 gaggtcaagc tgcaggagtc aggaactgaa catcttcaca agttatgata 101agtgaagttg tcctgcaagg cttctggcta gacttgagtg gattggatgg 151tagactgggt gaggcagacg cctgaacagg aatgagaagt tcaagggcag 201ggccacactg agtgtagaca agtcctccag cacagcctat atggagctca 251ctaggctgac atctgaggac tctgctgtct atttctgtgc tagaggggac 301tactataggc gctactttga cttgtggggc caagggacca cggtcaccgt 351ctcctcagga ggcggtggat cgggcggtgg cggctcgggt ggcggcggct 401ctgaaaatgt gctcacccag tctccagcaa tcatgtctgc atctccaggg 451gagagggtca ccatgacctg cagtgccagc tcaagtatac gttacatata 501ttggtaccaa cagaagcctg gatcctcccc cagactcctg atttatgaca 551catccaacgt ggctcctgga gtcccttttc gcttcagtgg cagtgggtct 601gggacctctt attctctcac aatcaaccga atggaggctg aggatgctgc 651cacttattac tgccaggagt ggagtggtta tccgtacacg ttcggagggg 701ggaccaagct ggagctgaaa gcgaaagaaa ccgctgctgc taaattcgaa 751cgccagcaca tggacagc

TABLE 2B Amino Acid Sequence for ScFv, SEQ. ID NO: 4   1EVKLQESGTE VVKPGASVKL SCKASGYIFT SYDIDWVRQT PEQGLEWIGW  51IFPGEGSTEY NEKFKGRATL SVDKSSSTAY MELTRLTSED SAVYFCARGD 101YYRRYFDLWG QGTTVTVSSG GGGSGGGGSG GGGSENVLTQ SPAIMSASPG 151ERVTMTCSAS SSIRYIYWYQ QKPGSSPRLL IYDTSNVAPG VPFRFSGSGS 201GTSYSLTINR MEAEDAATYY CQEWSGYPYT FGGGTKLELK AKETAAAKFE 251 RQHMDS

TABLE 3 Primers for PCR amplification of scFv(s) gene1. Primers for cloning of variable regions of heavy chain andlight chain of antibody (A) Primers for heavy chain (VH)Forward primer: 5'-GGCCCAGCCGGCCGAGGTCAAGCTGCAGGAGTCAGGA-3'(SEQ ID NO: 9) Reverse primer: 5'-CTCGGAACCTGAGGAGACGGTGACCGTGGTCCC-3'(SEQ ID NO: 10) (B) Primers for light chain (VL)Forward primer: 5'-TCCAAAGTCGACGAAAATGTGCTCACCCAGTCTCCA-3'(SEQ ID NO: 11) Reverse primer: 5'-AGCGGCCGCTTTCAGCTCCAGCTTGGTCCCCCC-3'(SEQ ID NO: 12)2. Primers for subcloning of ScFv(s) gene into pET-11a expression vector(A) Primers for heavy chain (VH) and linker amplificationForward primer: 5'-GTCAAGCTGCAGGAGTCAGGA-3' (SEQ ID NO: 13)Reverse primer: 5'-AGAGCCGCCGCCACCCGAGCCGCCACCGCCCGATCC (SEQ ID NO: 14)ACCGCCTCCTGAGGAGACGGTGACCGTGGT-3'(B) Primers for light chain (VL), linker and S-tag amplificationForward primer: 5'-GGAGGCGGTGGATCGGGCGGTGGCGGCTCGGGTGGC (SEQ ID NO: 15)GGCGGCTCTGAAAATGTGCTCACCCAGTCT-3'Reverse primer: 5'-AGTCAGGCTAGCTTAGCTGTCCATGTGCTGGCGTTCG (SEQ ID NO: 16)AATTTAGCAGCAGCGGTTTCTTTCGCTTTCAGCTCCAGCTT-3'

Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described inSambrook et al. (1989) Molecular Cloning, Second Edition, Cold SpringHarbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) MolecularCloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993)Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wu et al.(eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.)Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Oldand Primrose (1981) Principles of Gene Manipulation, University ofCalifornia Press, Berkeley; Schleif and Wensink (1982) Practical Methodsin Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRLPress, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic AcidHybridization, IRL Press, Oxford, UK; Setlow and Hollaender (1979)Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press,New York; Fitchen, et al. (1993) Annu Rev. Microbiol. 47:739-764;Tolstoshev, et al. (1993) in Genomic Research in Molecular Medicine andVirology, Academic Press; and Ausubel et al. (1992) Current Protocols inMolecular Biology, Greene/Wiley, New York, N.Y. Abbreviations andnomenclature, where employed, are deemed standard in the field andcommonly used in professional journals such as those cited herein.Antibody vaccines are described in Dillman R. O. (2001) Cancer Invest.19(8):833-841. Durrant L. G. et al. (2001) Int J. Cancer 1; 92(3):414-20and Bhattacharya-Chatterjee M, (2001) Curr. Opin. Mol. Ther. February;3(1):63-9 describe anti-idiotype antibodies. Many of the proceduresuseful for practicing the present invention, whether or not describedherein in detail, are well known to those skilled in the arts ofmolecular biology, biochemistry, immunology, and medicine.

Monoclonal, polyclonal antibodies, peptide-specific antibodies or singlechain recombinant antibodies and antigen binding fragments of any of theforegoing, specifically reacting with the ENOX2 isoform proteinsdescribed herein, may be made by methods known in the art. See e.g.,Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratories; and Goding (1986) Monoclonal Antibodies: Principlesand Practice, 2d ed., Academic Press, New York.

All references cited in the present application are incorporated byreference herein to the extent that there is no inconsistency with thepresent disclosure. Such references reflect the skill in the artsrelevant to the present invention.

The examples provided herein are for illustrative purposes, and are notintended to limit the scope of the invention as claimed herein. Anyvariations in the exemplified antibodies, epitopes, purificationmethods, diagnostic methods, preventative methods, treatment methods,and other methods which occur to the skilled artisan are intended tofall within the scope of the present invention.

What is claimed is:
 1. A single chain variable region ENOX2 antibodylinked to an alkaline phosphatase or other single step agent fordetection or imaging.
 2. The antibody of claim 1, wherein the singlechain variable region ENOX2 antibody comprises the polypeptide sequencesubstantially as shown in SEQ ID NO:5.
 3. The antibody of claim 1,wherein the single step agent for detection or imaging is enzymatic,chromagenic, chemiluminescent, magnetic or fluorescent.
 4. The antibodyof claim 1, wherein the single chain variable region ENOX2 antibody islinked to an S-tag that binds to a single step agent for detection orimaging.
 5. The antibody of claim 1, wherein the single chain variableregion ENOX2 antibody is coupled to a single step agent for detection orimaging.
 6. The antibody of claim 1, wherein the single chain variableregion ENOX2 antibody is chemically linked directly to an alkalinephosphatase.
 7. The antibody comprising the single chain variable regionantibody of claim 1 in aqueous solution at a concentration suitable foruse as an assay reagent.